Preventatives/remedies for skin aging

ABSTRACT

A composition for prophylaxis and therapy of dermal aging which comprises a substance having human leukocyte elastase inhibitory activity as an active ingredient.

TECHNICAL FIELD

[0001] This invention relates to a composition for prophylaxis andtherapy of dermal aging which comprises a substance having humanleukocyte elastase inhibitory activity as an active ingredient.

BACKGROUND ART

[0002] Aging of the skin is said to begin after the third decade oflife. Obvious signs of aging include decreases in the moistness, gloss,smoothness, tonus, etc. of the skin and an increased number of wrinkles.These are suspected to result from the morphological and functionalchanges of the organs and tissues making up the skin. Thus, aging isaccompanied by thinning of the epidermis and loss of oxytalan fiber inthe papillary layer of the dermis.

[0003] Dermal aging, epitomized by wrinkling of the skin, is a seriousbeauty problem for women but no satisfactory remedy has been availableto this day.

[0004] The present inventors discovered that a substance having humanleukocyte elastase inhibitory activity is effective in the preventionand treatment of dermal aging and has perfected this invention.

DISCLOSURE OF INVENTION

[0005] This invention is directed to a composition for prophylaxis andtherapy of dermal aging which comprises a substance having humanleukocyte elastase inhibitory activity as an active ingredient.

[0006] The substance having human leukocyte elastase inhibitory activitywhich can be used as the active ingredient of this composition forprophylaxis and therapy of dermal aging may be any substance that hashuman leukocyte elastase inhibitory activity. Furthermore, the substancehaving human leukocyte elastase activity which can be used in thisinvention includes not only substances which are direct inhibitors ofthat activity but also substances which inhibit leukocyte elastaseactivity indirectly through suppression of leukocyte infiltration andinhibition of elastase production. Thus, while many substances havingsuch activity are known, any novel substance that may have humanleukocyte elastase inhibitory activity can also be employed. Thefollowing is a partial listing of the preferred compounds in thiscategory. (1) WS7622A mono- or disulfate and pharmaceutically acceptablesalts thereof; among these, WS7622A disulfate ester disodium salt andWS7622A disulfate ester dipotassium salt are known substances which havethe following physicochemical properties (JP Kokai H4-279600).

[0007] WS7622A disulfate ester disodium salt (hereinafter sometimesreferred to briefly as FR134043):

[0008] Description: Colorless crystals

[0009] Solubility: Soluble: water, methanol

[0010] Insoluble: chloroform, n-hexane

[0011] Melting point: 257˜263° C. (decomp.) Optical rotation: [α]²³_(D)+37.5° (c=1, methanol) Molecular formula: C₄₇H₆₁N₉O₁₉S₂Na₂ Elementalanalysis: Calcd.: (for C₄₇H₆₁N₉O₁₉S₂Na₂.6H₂O): C, 44.30; H, 5.77; N,9.89; S, 5.03; Na, 3.61% Found: C, 44.98; H, 5.90; N, 10.06; S, 5.00;Na, 3.98% Molecular weight: FAB-MS m/z 1188 (M+Na)⁺

[0012] Thin-Layer Chromatography: TABLE 1 Stationary Developer phasesolvent Rf Silica gel CHCl₃—CH₃OH—H₂O 0.11 (Merck Art. 5715) (65:25:4)n-butanol-acetic 0.29 acid-water (4:2:1) Infrared absorption spectrum:υ^(KBr) _(max): 3360, 2960, 1735, 1660, 1640, 1530, 1500, 1380, 1250,1200, 1060, 1030, 940, 890 cm⁻¹ ¹H Nuclear magnetic resonance spectrum:(400 MHz, D₂O) δ 7.50 (1H, s) 7.27 (1H, s) 7.33-7.24 (3H, m) 6.94 (1H,q, J=7 Hz) 6.85 (2H, br d, J=8 Hz) 5.53 (1H, m) 5.37 (1H, m) 4.80 (1H,br s) 4.63-4.57 (2H, m) 4.53 (1H, m) 4.06 (1H, m) 3.99 (1H, d, J=10 Hz)3.56 (1H, br d, J=14 Hz) 3.46 (1H, m) 2.97 (3H, s) 2.97-2.88 (2H, m)2.72 (1H, m) 2.59 (1H, m) 2.51-2.38 (2H, m) 2.09-1.91 (4H, m) 1.82-1.60(3H, m) 1.77 (3H, d, J=7 Hz) 1.50 (3H, d, J=6.5 Hz) 1.40 (1H, m) 1.11(6H, d, J=7 Hz) 0.99 (3H, d, J=6.5 Hz) 0.97 (3H, d, J=6.5 Hz) ¹³CNuclear magnetic resonance spectrum: (100 MHz, D₂O) δ 183.6 (s) 177.9(s) 177.7 (s) 174.8 (s) 173.8 (s) 173.3 (s) 172.4 (s) 167.8 (s) 161.5(s) 145.5 (s) 144.9 (s) 139.6 (d) 139.0 (s) 137.0 (s) 136.0 (s) 132.3(d)×2 131.0 (d)×2 129.6 (d) 127.4 (d) 125.9 (d) 77.4 (d) 75.1 (d) 63.8(d) 62.7 (d) 59.1 (d) 55.9 (d) 54.9 (d) 51.9 (d) 41.9 (t) 37.2 (d) 36.9(t) 34.1 (q) 32.3 (d) 31.9 (t) 31.8 (t) 31.2 (t) 27.5 (t) 23.7 (t) 21.7(q) 21.4 (q)×2 21.3 (q) 21.1 (q) 15.5 (q)

[0013] Amino Acid Analysis:

[0014] WS7622A disulfate ester disodium salt (1 mg) was hydrolyzed with6 N-hydrochloric acid at 110° C. for 20 hours and, then, concentrated todryness under reduced pressure, and the residue was analyzed withHitachi 835 Automatic Amino Acid Analyzer. As the amino acids standardsolution, Wako Pure Chemical's Type H (Wako Code 013-08391) and Type B(016-08641) were used.

[0015] As a result, threonine, valine, phenylalanine, ornithine, ammoniaand several unknown ninhydrin-positive components were detected.

[0016] As a partial chemical structure of WS7622A disulfate esterdisodium salt, the following formula is proposed.

[0017] WS7622A disulfate ester dipotassium salt:

[0018] Description: Colorless amorphous powders

[0019] Solubility: Soluble: water, methanol

[0020] Insoluble: chloroform, n-hexane

[0021] Melting point: 230˜237° C. (decomp.) Optical rotation: [α]²³_(D)+34° (c=1, methanol) Molecular formula: C₄₇H₆₁N₉O₁₉S₂K₂ Elementalanalysis: Calcd.: (for C₄₇H₆₁N₉O₁₉S₂K₂.6H₂O ): C, 43.21; H, 5.63; N,9.65; S, 7.91; Na, 5.99% Found: C, 43.96; H, 5.44; N, 9.97; S, 5.09; Na,4.49% Molecular weight: FAB-MS m/z 1236 (M+K)⁺

[0022] Thin-Layer Chromatography: TABLE 2 Stationary Developer phasesolvent Rf Silica gel CHCl₃—CH₃OH—H₂O 0.13 (Merck Art. 5715) Infraredabsorption spectrum: υ^(KBr) _(max): 3360, 2960, 1735, 1660, 1640, 1530,1500, 1405, 1380, 1250, 1200, 1050, 1030, 940, 890 cm⁻¹ ¹H Nuclearmagnetic resonance spectrum: (400 MHz, D₂O) δ: 7.52 (1H, s) 7.28 (1H, s)7.34-7.25 (3H, m) 6.96 (1H, q, J=7 Hz) 6.87 (2H, br d, J=8 Hz) 5.56 (1H,m) 5.40 (1H, m) 4.84 (1H, br s) 4.70-4.55 (3H, m) 4.10 (1H, m) 4.03 (1H,m) 3.60 (1H, br d, J=14 Hz) 3.50 (1H, m) 3.00 (3H, s) 3.00-2.85 (2H, m)2.76 (1H, m) 2.62 (1H, m) 2.55-2.40 (2H, m) 2.12-1.95 (4H, m) 1.90-1.65(3H, m) 1.79 (3H, d, J=7 Hz) 1.53 (3H, d, J=6.5 Hz) 1.45 (1H, m) 1.14(6H, d, J=7 Hz) 1.02 (3H, d, J=6.5 Hz) 1.00 (3H, d, J=6.5 Hz)

[0023] Amino Acid Analysis:

[0024] WS7622A disulfate ester dipotassium salt (1 mg) was hydrolyzedwith 6 N-hydrochloric acid (1 ml) at 110° C. for 20 hours and, then,concentrated to dryness under reduced pressure, and the residue wasanalyzed with Hitachi 835 Automatic Amino Acid Analyzer. As the aminoacids standard solution, Wako Pure Chemical's Type H (Wako Code013-08391) and Type B (016-08641) were used.

[0025] As a result, threonine, valine, phenylalanine, ornithine, ammoniaand several unknown ninhydrin-positive components were detected.

[0026] As a partial chemical structure of WS7622A disulfate esterdipotassium salt, the following formula is proposed.

[0027] As the pharmaceutically acceptable salt of WS7622A mono- ordisulfate ester, there can be mentioned the mono- or di-salts withinorganic or organic bases, such as alkali metal salts (e.g. sodiumsalt, potassium salt, etc.), alkaline earth metal salts (e.g. calciumsalt etc.), ammonium salt, ethanolamine salt, triethylamine salt,dicyclohexylamine salt and pyridine salt, among others.

[0028] Substance WS7622A which is a starting compound for synthesis ofsaid WS7622A mono- or disulfate ester also has human leukocyte elastaseinhibitory activity and can, therefore, be used as a prophylactic andtherapeutic agent for wrinkles. This substance is known to have thefollowing physicochemical properties (JP Kokai H3-218387, JP KokaiH4-279600).

[0029] Physicochemical properties of Substance WS7622A:

[0030] Description: colorless prisms

[0031] Acidity-alkalinity: acidic

[0032] Color reactions:

[0033] Positive: cerium sulfate reaction, iodine vapor reaction, ferricchloride reaction

[0034] Negative: ninhydrin reaction, Molisch reaction, Dragendorffreaction

[0035] Solubility:

[0036] Soluble: methanol, ethanol, n-butanol

[0037] Slightly soluble: chloroform, acetone, ethyl acetate

[0038] Insoluble: water, n-hexane

[0039] Thin-layer chromatography (TLC):

[0040] Chloroform-methanol (5:1, v/v)

[0041] Rf 0.51

[0042] Acetone-methanol (10:1)

[0043] Rf 0.62

[0044] (Kieselgel 60F₂₅₄ silica gel plate, Merck)

[0045] Melting point: 250˜252° C. (decomp.) Optical rotation: [α]²³_(D)+36° (c=1, methanol) UV absorption spectrum: λ^(MeOH) _(max) 287 nm(ε=3600) λ^(MeOH—HC1) _(max) 287 nm λ^(MeOH—NaOH) _(max) 298 nmMolecular formula: C₄₇H₆₃N₉O₁₃ Elemental analysis: Calcd.: (forC₄₇H₆₃N₉O₁₃.2H₂O): C, 56.56; H, 6.77; N, 12.63% Found: C, 56.65; H,6.62; N, 12.27% Molecular weight: FAB-MS m/z 984 (M+Na)⁺Infraredabsorption spectrum: υ^(KBr) _(max): 3400, 3300, 3060, 2980, 2940, 1735,1710, 1690, 1670, 1660, 1640, 1540, 1520, 1470, 1380, 1330, 1300, 1260,1220, 1200, 1160, 1130, 1090, 1000, 980, 940, 920 cm⁻¹ ¹H Nuclearmagnetic resonance spectrum: (400 MHz, CD₃OD) δ: 7.22-7.09 (3H, m)6.88-6.77 (3H, m) 6.74 (1H, s) 6.46 (1H, s) 5.46 (1H, m) 5.18 (1H, s)4.85 (1H, s) 4.77 (1H, m) 4.65 (1H, m) 4.50 (1H, m) 3.96 (1H, m) 3.91(1H, d, J=9 Hz) 3.60-3.47 (2H, m) 3.03 (1H, m) 2.90 (3H, s) 2.86 (1H, m)2.59-2.49 (2H, m) 2.39 (1H, m) 2.29-2.16 (2H, m) 2.00 (1H, m) 1.84 (1H,m) 1.74 (3H, d, J=6 Hz) 1.72-1.53 (4H, m) 1.44 (3H, d, J=6 Hz) 1.12 (1H,m) 1.10 (6H, d, J=6 Hz) 0.99 (3H, d, J=6 Hz) 0.94 (3H, d, J=6 Hz) ¹³CNuclear magnetic resonance spectrum: (100 MHz, CD₃OD) δ 179.7 (s) 176.3(s) 174.7 (s) 173.3 (s) 172.4 (s) 171.4 (s) 170.3 (s) 165.8 (s) 160.2(s) 145.7 (s) 145.6 (s) 137.5 (s) 134.0 (d) 131.4 (s) 130.6 (d)×2 129.8(s) 129.1 (d)×2 129.1 (s) 127.6 (d) 119.1 (d) 118.0 (d) 76.0 (d) 73.4(d) 63.1 (d) 61.4 (d) 57.1 (d) 53.6 (d) 52.7 (d) 50.5 (d) 39.9 (t) 36.1(t) 35.8 (d) 31.8 (q) 31.0 (t) 30.8 (d) 29.9 (t) 29.7 (t) 25.2 (t) 22.3(t) 20.2 (q) 20.0 (q)×2 19.7 (q) 19.5 (q) 13.3 (q)

[0046] Amino Acid Analysis:

[0047] WS7622A (1 mg) was hydrolyzed with 6 N-hydrochloric acid (1 m) at110° C. for 20 hours and concentrated to dryness under reduced pressure,and the residue was analyzed with Hitachi 835 Automatic Amino AcidAnalyzer. As the amino acids standard solution, Wako Pure Chemical'sType H (Wako Code 013-08391) and Type B (016-08641) were used.

[0048] As a result, threonine, valine, phenylalanine, ornithine, ammoniaand several unknown ninhydrin-positive components were detected.

[0049] As a partial chemical structure of WS7622A, the following formulais proposed.

[0050] The salt of Substance WS7622A includes salts with inorganic ororganic bases, such as alkali metal salts (e.g. sodium salt, potassiumsalt, etc.), alkaline earth metal salts (e.g. calcium salt etc. ),ammonium salt, ethanolamine salt, triethylamine salt anddicyclohexylamine salt, among others.

[0051] Substances WS7622B, C and D and derivatives thereof (JP KokaiH3-218387) , all of which have human leukocyte elastase inhibitoryactivity as well can also be used as prophylactic and therapeutic agentsfor wrinkles.

[0052] The above-mentioned Substance WS7622A (as well as SubstancesWS7622B, C and D) can be produced by growing Streptomycesresistomycificus No. 7622, which strain has been deposited with NationalInstitute of Bioscience and Human Technology, one of the internationalculture collections under Budapest Treaty, with the accession number ofFERM BP-2306 assigned.

[0053] (2) A trifluoromethylketone derivative of the following formulaand its pharmaceutically acceptable salt:

[0054] [wherein R¹ represents a lower alkyl group having 1 or 2substituents selected from among carboxy, esterified carboxy anddi-lower alkylcarbamoyl; a phenyl (lower) alkyl group which mayoptionally have halogen, amino or nitro on the phenyl moiety thereof andcarboxy or esterified carboxy on the alkyl moiety thereof; a halophenylgroup; a morpholino group; or a morpholino (lower) alkyl group; R² andR³ each represents a lower alkyl group; X represents— or —NH—;

[0055] (3) a trifluoromethylketone derivative of the following formulaand its pharmaceutically acceptable salt:

[0056] (wherein R¹˜R³ have the same meanings as defined above (2)).

[0057] (4) 3 (RS)-[[4-(carboxymethylaminocarbonyl)phenyl-carbonyl]-L-valyl-L-prolyl]amino-1,1,1-trifluoro-4-methyl-2-oxopentaneor its sodium salt (this sodium salt will sometimes be referred tobriefly as FK706).

[0058] The compounds mentioned in the above paragraphs (2)˜(4) are knowncompounds as described in JP Kokai H4-297446, for instance. As thepharmaceutically acceptable salts of said compounds (2)˜(3) , there canbe mentioned the respective salts with organic or inorganic bases, suchas alkali metal salts (e.g. sodium salts, potassium salts, etc.),alkaline earth metal salts (e.g. calcium salts etc.), ammonium salts,ethanolamine salts, triethylamine salts, dicyclo-hexylamine salts, etc., methanesulfonates, and organic or inorganic acid addition salts suchas hydrochlorides, sulfates, nitrates, phosphates and so forth.

[0059] The preferred examples relevant to the various definitions givenhereinbefore are now set forth in detail. The term “lower” means 1˜6carbon atoms unless otherwise specified. As the preferred examples of“halogen”, there can be mentioned fluoro, chloro, bromo and iodo. Thepreferred “lower alkyl group” includes residues of straight-chain orbranched-chain alkanes containing 1˜6 carbon atoms, such as methyl,ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, neopentyland hexyl, preferably those having 1˜4 carbon atoms. The preferred“esterified carboxy” includes, among others, alkyl esters, i.e.alkoxycarbonyl groups such as lower alkoxycarbonyl (e.g.methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,tert-butoxycarbonyl, etc.), and phenyl (lower) alkyl esters, i.e. phenyl(lower) alkoxycarbonyl groups such as benzyloxycarbonyl etc., andbenzoyl (lower) alkyl esters, i.e. benzoyl (lower) alkoxycarbonyl groupssuch as benzoylmethoxycarbonyl and so forth.

[0060] The preferred “lower alkylene group” includes but is not limitedto methylene, ethylene, propylene and isopropylene. The preferred“di-lower-alkylcarbamoyl group” includes N,N-dimethylcarbamoyl andN,N-diethylcarbamoyl, among others.

[0061] (5) Substance FR901451 having the following physicochemicalproperties and its pharmaceutically acceptable salt:

[0062] Description: white powders

[0063] Color reactions:

[0064] Positive: cerium sulfate, iodine vapor, Ehrlich, and ninhydrinreactions

[0065] Negative: Molisch reaction

[0066] Solubility:

[0067] Soluble: water, methanol, dimethyl sulfoxide

[0068] Hardly soluble: acetate

[0069] Insoluble: ethyl acetate

[0070] Melting point: 243˜245° C. (decomp.) Optical rotation: [α]²³_(D)−15° (c=0.65, H₂O) Ultraviolet absorption spectrum: λ^(MeOH) _(max)nm(ε): 275 (4300), 281 (4500), 290 (3900) Molecular formula:C₆₀H₇₉N₁₃O₁₈ Elemental analysis: Calcd.: (for C₆₀H₇₉N₁₃ O₁₈.10H₂O): C,49.68; H, 6.88; N, 12.55 Found: C, 49.95; H, 6.28; N, 12.42 Molecularweight: FAB-MS m/z 1270 (M+H)⁺

[0071] Thin-Layer Chromatography: TABLE 3 Stationary Developer phasesolvent Rf Silica gel CHCl₃:MeOH:NH₄OH 0.60 (Merck) (15:11:5) RP-18 70%aqueous 0.32 (Merck) methanol FT-IR spectrum: υ^(KBr) _(max): 3390,3070, 2970, 2880, 1740, 1660, 1530, 1450, 1410, 1380, 1350, 1250, 1190,1110, 1080, 1010, 750, 700, 670, 660, 620, 600 cm⁻¹ ¹H Nuclear magneticresonance spectrum: (400 MHz, D₂O) δ: 7.70 (1H, d, J=7 Hz) 7.52 (1H, d,J=7.5 Hz) 7.44-7.23 (7H, m) 7.22 (1H, s) 5.59 (1H, q, J=7 Hz) 4.94 (1H,t, J=4.5 Hz) 4.85-4.74 (3H, m) 4.58 (1H, dd, J=6 Hz, 10 Hz) 4.45-4.35(3H, m) 4.30 (1H, dd, J=4 Hz, 7 Hz) 4.07 (1H, m) 3.99 (1H, dd, J=10 Hz,4.5 Hz) 3.66-3.50 (3H, m) 3.44-3.25 (4H, m) 3.16-2.93 (4H, m) 2.87 (1H,d, J=18 Hz) 2.80-2.68 (2H, m) 2.56-2.48 (2H, m) 2.08 (1H, dd, J=16 Hz, 4Hz) 1.87-1.53 (9H, m) 1.43 (3H, d, J=7 Hz) 1.30 (3H, d, J=6.5 Hz)1.45-1.17 (4H, m) 0.95 (3H, d, J=6 Hz) 0.84 (3H, d, J=6 Hz) ¹³C Nuclearmagnetic resonance spectrum: (100 MHz, D₂O) δ 177.2 (s) 130.0 (d)×2 56.0(d) 31.4 (t) 176.5 (s) 129.8 (d)×2 54.1 (d) 28.8 (t) 174.6 (s) 128.5 (d)53.8 (d) 26.6 (t) 174.2 (s) 127.8 (s) 53.2 (d) 25.1 (d) 174.0 (s) 125.5(d) 53.1 (d) 23.2 (q) 173.2 (s) 123.2 (d) 52.9 (d) 23.2 (t) 173.0 (s)120.9 (d) 52.8 (d) 23.1 (t) 172.8 (s) 118.7 (d) 49.5 (d) 20.8 (q) 172.6(s) 113.1 (d) 48.6 (t) 19.4 (q) 172.5 (s) 108.8 (s) 40.1 (t) 18.3 (q)172.1 (s) 73.3 (d) 39.6 (t) 171.7 (s) 69.7 (d) 39.4 (t) 171.4 (s) 64.3(t) 38.9 (t) 170.3 (s) 62.1 (d) 35.3 (t) 137.2 (s) 60.9 (d) 34.8 (t)136.0 (s) 57.1 (d) 31.7 (t)

[0072] The above Substance FR901451 is known as the substance elaboratedby Substance FR901451-producing strains of organisms belonging to thegenus Flexibacter (e.g. WO93/02203). Flexibacter sp. No. 758, which isamong such producer strains, has been deposited with National Instituteof Bioscience and Human Technology, an international culture collectionunder Budapest Treaty, with the accession number of FERM BP-3420assigned.

[0073] The pharmaceutically acceptable salt of the above SubstanceFR901451 includes the same kinds of salts as mentioned for thepharmaceutically acceptable salts of the compounds (2)˜(3).

[0074] In addition to the foregoing substances, the following can bementioned as examples of the substance having elastase inhibitoryactivity: α1-antitrypsin, SLP1 (secretory leukocyte protease inhibitor)[American Review of Respiratory Diease Vol. 147, 1993, P442-446],urinastatin, colchicine, erythromycin, clarithromycin, ICI200, 880,ONO-8046 [American Journal of Respiratory and Critical Care MedicineVol. 153, P391-397], anti-elastase antibodies, and so forth.

[0075] For the purposes of this invention, a substance having humanleukocyte elastase inhibitory activity is efficacious and can beadministered for the prevention and treatment of dermal aging ingeneral. More particularly, it can be indicated for the prevention andtreatment of decreases in moistfulness, sheen, smoothness, and tonus ofthe skin and even increased wrinkling, or the prevention and treatmentof skin flaccidity. These signs of dermal aging are suspected to arisefrom morphological and functional changes of the organs and tissuesmaking up the skin, and actually, thinning of the horny layer of theepidermis and loss of oxytalan fiber in the papillary layer of thecorium are noted.

[0076] The signs of dermal aging being as mentioned above, the efficacyof a substance having human leukocyte elastase inhibitory activity isparticularly pronounced for the elimination or diminution of wrinkles orprevention of increased wrinkling, improvement of skin texture(fineness, handle) and amelioration of the shade of the skin (shadowycomplexion).

[0077] Furthermore, said substances are efficacious for promotingneogenesis of oxytalan fiber in the region of dermal papillae andneogenesis of collagen fibrils in the dermis immediately beneath theepidermis, and for increasing the thickness of the epidermis, amongothers.

[0078] In addition, substances having human leukocyte elastaseinhibitory activity are efficacious in various skin diseases such asscleroderma, elephantiasis, scars, steroid-induced thinning of the skin,keloids, pressure sores, wounds, refractory ulcers, psoriasis, spots,freckles, senile plaques, rough skin and pityriasis.

[0079] The following is an example of experimentation relevance to thisinvention.

[0080] Object of Experiment:

[0081] The therapeutic efficacy of neutrophil (leukocyte) elastaseinhibitors in “dermal aging” was evaluated in hairless dogs.

[0082] Experimental Animals:

[0083] Two adult (old) experimental hairless dogs constructed bycross-breeding of a Mexican hairless dog and a beagle dog were used. Olddogs presenting with age-associated fine “wrinkles”, not observed inyoung dogs, on the body surface were used as subject animals.

[0084] No. 8807 (10 yr old, male)

[0085] No. 8808 (10 yr old, female)

[0086] Investigational Drugs:

[0087] 1) FK706·0.2%

[0088] 2) FK706·0.02%

[0089] 3) FR134043·0.2%

[0090] 4) FR134043·0.02%

[0091] 5) Polyethylene glycol (PEG) (solvent control) (PEG was used asthe solvent for drugs 1˜4)

[0092] Administration:

[0093] Each drug was applied in one location (a total of 5 locations) onthe back (5×5 cm) of each dog. The drug was applied in a dosing volumeof about 4 μL/cm² once daily (except on Saturdays, Sundays and holidays)for 3 months.

[0094] Evaluation Items:

[0095] 1) Skin condition: Gross observation and observation with a videomacroscope

[0096] 2) Histology: Observation of histological changes in biopsysamples by H-E (hematoxylin-eosin) stain (general staining), vanGieson's stain (staining of collagen fiber) and Weigert's stain(staining of elastin fiber)

[0097] 3) Skin thickness: Using H-E stained tissue samples, changes inthickness of the epidermis (excluding the horny layer) were studied(only the location treated with FK706 0.2%, which showed a markedimprovement in skin condition, was evaluated)

[0098] Results:

[0099] 1) Skin Condition (Remission of Wrinkles)

[0100] (Gross Observation)

[0101] No. 8807: In the elimination or remission of wrinkles, the orderof efficacy was FK706·0.1%>FK706·0.02%>FR134043·0.2%=FR134043·0.02%.This finding of remission was accompanied by improvements in skintexture (fineness, handle) and paralleled depigmentation of the skin(change to fair˜rosy skin).

[0102] No. 8808: The order of efficacy wasFK706·0.2%>FK706·0.02%>FR134043·0.2%=FR134043·0.02%.

[0103] In neither dog was found a side effect.

[0104] (Observation with a Video Macroscope)

[0105] The findings were substantially identical to the results of grossobservation.

[0106] No. 8807: The order of efficacy wasFK706·0.2%>FK706·0.02%>FR134043·0.2%>FR134043·0.02%

[0107] No. 8808: The order of efficacy wasFK706·0.2%=FK706·0.02%>FR134043·0.2%=FR134043·0.02%.

[0108] 2) Histological Findings

[0109] Van Gieson's Stain:

[0110] Neogenesis of a thin layer of collagen fiber was found across theboundary between the epidermis and dermis in the case of FK706·0.2% (DogNo. 8807)

[0111] Weigert's Strain:

[0112] Growth of elastin fibrils (suspected to be oxytalan fiber whichis said to disappear as increasing age) was found across the boundarybetween the epidermis and dermis (Dog No. 8807).

[0113] 3) Evaluation of Skin Thickness

[0114] The thickness of the epidermis was increased significantly(p<0.05 in both Dog 8807 and Dog 8808).

[0115] No. 8807: 17.0±1.9 μm→26.8±5.9 μm (mean±SD)

[0116] No. 8808: 24.6±3.6 μm→42.0±9.0 μm

[0117] The composition for prophylaxis and therapy of dermal aging asprovided by this invention is usually applied in the form of apreparation for external application (e.g. lotion, ointment, patch,liniment, aerosol, suspension, emulsion, etc.) or an external powder(e.g. an enzyme facial cleanser) but may also be used in theconventional pharmaceutical dosage forms such as powders, fine granules,granules, tablets, sugar-coated tablets, parenteral preparations,inhalants, microcapsules, capsules, suppositories, solutions, syrups andso on. Furthermore, it can be used in such formulations as facialcleansers, facial wash-off/emolient preparations, and bath preparations.Where necessary, a diluent or disintegrator (e.g. sucrose, lactose,starch, crystalline cellulose, low-substitution-degreehydroxypropylcellulose, synthetic aluminum silicate, etc.), a binder(e.g. cellulose, methylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, polypropylpyrrolidone,polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, etc.), acoloring agent, a sweetener and a lubricant (e.g. magnesium stearate),among others, can be additionally dispersed in such formulations.

[0118] The level of use of the composition for prophylaxis and therapyof dermal aging according to this invention depends on the species ofsubstance used, symptoms and other factors but, generally speaking, therecommended dose of an external dosage form, in terms of theconcentration of the substance having human leukocyte elastaseinhibitory activity or a pharmaceutically acceptable salt thereof, canbe judiciously selected from the range of about 0.001˜20%, preferablyabout 0.01˜10%.

1. A composition for prophylaxis and therapy of dermal aging whichcomprises a substance having human leukocyte elastase inhibitoryactivity as an active ingredient.
 2. A composition for prophylaxis andtherapy of wrinkles which comprises WS7622A mono- or disulfate ester ora pharmaceutically acceptable salt thereof as an active ingredient.
 3. Acomposition for prophylaxis and therapy of dermal aging which comprisesa trifluoromethylketone derivative of the following formula or apharmaceutically acceptable salt thereof as an active ingredient.

[wherein R¹ represents a lower alkyl group having 1 or 2 substituentsselected from among carboxy, esterified carboxy and di-loweralkylcarbamoyl; a phenyl (lower) alkyl group which may optionally havehalogen, amino or nitro on the phenyl moiety thereof and carboxy oresterified carboxy on the alkyl moiety thereof; a halophenyl group; amorpholino group; or a morpholino (lower) alkyl group; R² and R³ eachrepresents a lower alkyl group; X represents— or —NH—;


4. A composition for prophylaxis and therapy of wrinkles which comprisesa trifluoromethylketone derivative of the following formula or apharmaceutically acceptable salt thereof as an active ingredient.

[wherein R¹ represents a lower alkyl group having 1 or 2 substituentsselected from among carboxy, esterified carboxy and di-loweralkylcarbamoyl; a phenyl (lower) alkyl group which may optionally havehalogen, amino or nitro on the phenyl moiety thereof and carboxy oresterified carboxy on the alkyl moiety thereof; a halophenyl group; amorpholino group; or a morpholino (lower) alkyl group; R² and R³ eachrepresents a lower alkyl group; X represents— or —NH—;


5. The composition for prophylaxis and therapy of dermal aging asclaimed in claim 1 wherein the substance having human leukocyte elastaseinhibitory activity is Substance FR134043.
 6. The composition forprophylaxis and therapy of wrinkles as claimed in claim 2 wherein thesubstance having human leukocyte elastase inhibitory activity isSubstance FR134043.
 7. The composition for prophylaxis and therapy ofdermal aging as claimed in claim 1 wherein the substance having humanleukocyte elastase inhibitory activity is Substance FK706.
 8. Thecomposition for prophylaxis and therapy of wrinkles as claimed in claim4 wherein the substance having human leukocyte elastase inhibitoryactivity is Substance FK706.
 9. A method for prophylaxis and therapy ofdermal aging which comprises administering a substance having humanleukocyte elastase inhibitory activity to a patient for the preventionor treatment of dermal aging.
 10. Use of a substance having humanleukocyte elastase inhibitory activity for the production of apharmaceutical composition for the prophylaxis and therapy of dermalaging.